AurkA nuclear localization is promoted by TPX2 and counteracted by protein degradation

This study shows that TPX2 co-overexpression in non-transformed human cells induces AurkA nuclear accumulation and promotes its non-mitotic oncogenic functions.


Introduction
AurkA is a member of the Aurora family of serine/threonine kinases (1,2). AurkA is a well-known mitotic regulator, with key roles in mitotic entry, centrosome maturation, and spindle organization through phosphorylation of several substrates, such as Cdk1 and Plk1 (3,4,5,6). AurkA levels are cell cycle-regulated; they increase in the late S and G2 phases, peak in mitosis, and rapidly decrease at the mitotic exit when the kinase is degraded in a proteasome-dependent manner (7,8). Interaction with its major activator TPX2 is required for complete AurkA activation; TPX2 is also a regulator of AurkA stability and localization at spindle microtubules (9,10,11,12). AURKA overexpression is frequently observed in many cancer types (13,14) and targeting the kinase is studied as an anti-cancer therapeutic approach (13,15,16,17). Inhibitors of AurkA kinase activity are under evaluation in clinical trials, but moderate effects and partial specificity of action are observed (14,15,16,17,18). In recent years, evidence of AurkA non-mitotic roles, also in the G0/G1 phases, is emerging, for example, involvement in neurite outgrowth in post-mitotic neurons, primary cilium disassembly, DNA replication, and regulation of mitochondrial morphology and dynamics (19,20,21). We recently reviewed published evidence, integrated with data mining searches, correlating the oncogenic potential of AurkA with its increased localization at the interphase nucleus in tumors (solid and hematological) and hence with non-mitotic functions (22). In particular, in breast cancer AurkA nuclear localization has been proposed as a prognostic marker for poor survival (23) and has been shown to associate with transcriptional up-regulation (24,25) and stabilization (26) of known oncogenes, such as FOXM1 and myc family members. A role of nuclear AurkA in activation of hypoxia transcriptional programs has been also recently reported in breast cancer, a condition that drives cell migration, morphological changes, and increased stemness, thus determining dissemination and metastases at other organs (27). Interestingly, some of the described roles of nuclear AurkA are reported as kinase-independent (reviewed in reference 22). These observations strengthen the interest in nuclear roles of AurkA in cancer development and progression. Still, how AurkA nuclear localization is regulated under physiological and pathological conditions has been so far poorly investigated.
In the present study, we investigated the nuclear localization of AurkA to clarify the molecular mechanisms through which it is regulated. We analyzed non-mitotic AurkA localization in a 1 non-transformed cellular background and observed how this localization is influenced by the cell cycle phase and the nuclear export, whereas it is not affected by its catalytic activity. By AURKA overexpression studies, we then uncovered that AurkA increased levels alone are not sufficient to determine increased AurkA localization. Gene expression analyses revealed a strong cooverexpression of AURKA, TPX2, and exportin-2 CSE1L in tumors. Interestingly, when AURKA is overexpressed in non-transformed cells, its nuclear localization is increased upon TPX2 cooverexpression, or after proteasome inhibition, indicating involvement of AurkA protein stability. This TPX2 role is independent of its AurkA-activating function. Finally, we show that TPX2 contribution to AurkA nuclear localization has functional consequences in MCF10A cells on tumor-related AurkA roles. Together, our results suggest that AURKA/TPX2 co-overexpression in cancer has an impact not only on mitotic but also on interphasic nuclear AurkA oncogenic functions.

Results
Cell cycle phase and protein export influence AurkA nuclear levels To investigate how nuclear localization of AurkA is regulated, we first analyzed endogenous AurkA by immunofluorescence (IF) in three] representative experiment is shown); the percentage of cells in the G2 and M phases is indicated in the table below (mean ± SD, three independent experiments). Histograms represent the percentage of interphases with nuclear AurkA localization in control cultures and after the indicated treatments (at least 450 cells; three independent experiments). (E) Representative images, from a time-lapse experiment, of the dynamic accumulation of endogenous AurkA (Venustagged, hTERT RPE-1 cells) before mitotic entry (10 ≤ n ≤ 49 at different timepoints, because individual trajectories in this experiment were not synchronized; two experiments). The first observable frame in mitosis is set as time 0 (images and graph), and a time interval of 240 min before mitotic entry is analyzed for mean nuclear fluorescence (red plot; a.u., arbitrary units) and nucleo/cytoplasmic ratios (blue plot). In the shown example, nuclear accumulation is observable from t = −90 min. (F) Representative IF images for AurkA and PCNA staining in the control culture and upon indicated treatments. Histograms represent the percentage of cells displaying nuclear AurkA that are also positive for PCNA staining (arrowed in the control panel), in the indicated conditions (at least 150 cells; three independent experiments). Error bars: SD; ns: not significant; **P < 0.001; and ***P < 0.0001, chi-squared test. Scale bars: 10 μm (A, B, C) or 20 μm (E, F). non-transformed hTERT RPE-1 cells (Fig 1). AurkA staining is evident in about one-third of interphases: a fraction of cells only displays a centrosomal signal, whereas AurkA is nuclear (either uniformly diffused between the nucleus and the cytoplasm or nuclearenriched; see Fig 1A) in 20% of all interphases. Cells with nuclear AurkA display two spots of Cep170 (Fig 1B), characteristic of the G2 phase, and an intact nuclear envelope as assessed by lamin B1 staining ( Fig 1C). Indeed, the percentage of cells with nuclear AurkA is consistent with the fraction of asynchronous hTERT RPE-1 cell population being in the G2 or M phases, and enriching the population in G2 by treatment with the Cdk1 inhibitor RO3306 induced a parallel increase in interphases with nuclear AurkA (Fig 1D). Video recording of hTERT RPE-1 cells expressing AURKA endogenously tagged with Venus (20) shows that an increase in AurkA nuclear levels begins about 90 min before mitotic entry, paralleled by a slight increase in the nucleo/cytoplasmic ratio ( Fig 1E). Together, these observations indicate that in non-transformed cells AurkA nuclear localization occurs in G2. Import/export processes regulating AurkA localization in interphase are not well clarified. Rannou et al (28) reported an increase in nuclear AurkA-exogenously expressed-after export inhibition. To investigate the relevance of active export of endogenous AurkA, we treated hTERT RPE-1 cells with leptomycin B (LMB), an inhibitor of the export receptor CRM1. Under these conditions, the cell cycle is not altered (FACS panels and table, Fig 1D) and AurkA is nuclear in about 40% of cells compared with 20% in control cultures ( Fig 1D). These data suggest that active export may modulate the nuclear localization of the kinase. AurkA nuclear localization was not increased when we combined RO3306 with LMB treatments, compared to treatment with RO3306 alone (Fig 1D), suggesting that the export process is relevant for AurkA localization in a time window preceding G2. To confirm this hypothesis, cells were co-stained with the S-phase marker PCNA: results show that after LMB treatment, more than 40% of cells with nuclear AurkA are PCNA-positive ( Fig  1F), compared with 20% in control cells. This increased association was not observed when cells were treated with RO3306, either alone or in combination with LMB, due to the enrichment of cells in the G2 phase. Indeed, the significant decrease in S-phase PCNA-positive cells after RO3306 treatment ( Fig S1A) explains the lack of additive effects of the combined treatments (RO3306 + LMB) on AurkA nuclear localization (Fig 1D).
These results indicate that AurkA localization in the nucleus under physiological conditions strongly correlates with the G2 phase, and nuclear export contributes to limit AurkA nuclear accumulation in previous cell cycle phases.

AURKA overexpression in non-transformed cells is not sufficient to yield its nuclear enrichment
To evaluate whether AURKA overexpression is sufficient to yield high levels of nuclear AurkA, which have been reported as oncogenic in cancer cells, we used an hTERT RPE-1 cell line that we have generated for stable and inducible expression of exogenous myctagged AurkA (Fig 2A (29)). In this cell line, after doxycycline (dox) induction, <60% of interphases are positive for AurkA staining (compared with 30% of hTERT RPE-1 cells staining positive for endogenous AurkA). Nonetheless, only about 15% of all cells displayed AurkA nuclear localization (Fig 2B), a percentage comparable to control cultures ( Fig 1D). Instead, a peculiar distribution rarely observed for endogenous AurkA in hTERT RPE-1 control cells was evident; that is, in about 50% of interphases, AurkA localized to the cytoplasm and appeared excluded from nuclei (Fig 2A and B). Inhibition of protein export by LMB determines a small, but significant, increase in cells with nuclear accumulation of AurkA ( Fig  2B), indicating only a partial involvement of nuclear export in AurkA nuclear exclusion under overexpression conditions. Unexpectedly, the percentage of AurkA-positive cells decreased after LMB treatment (Fig 2B, left histograms); consistently, the overall amount of AurkA was reduced (Western blotting [WB] in Fig 2C). These observations suggest that inhibiting export from the nucleus, and entrapping AurkA therein, determines increased AurkA degradation, influencing the overall amount of the protein. To directly address this, we combined LMB with MG132 treatment to simultaneously inhibit nuclear export and proteasome degradation activity. Under these conditions, AurkA levels are higher (Fig 2C, compare the last two lanes) and cells with AurkA nuclear localization increase (90% of AurkA-positive cells) with an evident nuclear enrichment ( Fig 2B). Interestingly, MG132 treatment alone was able to yield the same effect (Fig 2B), indicating that AurkA nuclear stabilization has a dominant effect over protein export. Comparable results were obtained by inhibiting the proteasome with epoxomicin (Fig S1B), suggesting that nuclear accumulation of AurkA is normally counteracted by protein degradation. FACS analyses indicated that neither MG132 nor epoxomicin treatment yielded major changes in cell cycle progression (Fig S1C). Given that AurkA is an APC/C-Fzr1 target (8,30,31) and Fzr1 is reported to be nuclear in interphase (32,33), we investigated whether it is involved in AurkA nuclear degradation. Indeed, combined treatment with APC/C inhibitors Apcin and proTAME (34) in the AurkA-overexpressing cell line yielded a significant increase in cells with nuclear AurkA (45% compared with 15% in DMSO-treated cultures) and a corresponding decrease in AurkA nuclear-excluded cells ( Fig 2D). To investigate whether this was a direct effect on the kinase, we assayed the localization of a non-degradable AurkA-deleted version, AurkAΔ67-Venus, lacking the A-box described as critical for APC/C-Fzr1 recognition (35). Nucleo/cytoplasmic ratio fluorescence measures after transient transfection in U2OS cells indicated that this deleted version is indeed nuclear-enriched compared with WT AurkA-Venus ( Fig 2E).
In conclusion, we observed that overexpression of AURKA is not sufficient to increase nuclear localization of the kinase, which is negatively modulated by protein export and to a larger extent by protein degradation, suggesting that increased nuclear localization of AurkA in cancer cells depends on altered interphase regulatory mechanisms.

AURKA, TPX2, and CSE1L are co-overexpressed in cancer
To investigate altered mechanisms that may be linked to the nuclear enrichment of AurkA in cancer, we first carried out an analysis on RNA-sequencing data coming from TCGA and GTEx cancer consortium projects, mining for genes encoding proteins involved in nucleus/cytoplasm shuttling with expression profiles in tumors similar to AURKA. To this aim, we selected from gene ontology (GO) all the genes associated with import/export from the nucleus and we computed, for each one, the pairwise expression correlation to AURKA, using Pearson's correlation analysis. Strikingly, we found a high correlation between exportin-2 (also known as chromosome segregation 1 like, CSE1L) and AURKA in multiple cancer types, with breast invasive carcinoma-where nuclear AurkA has been described (24, 25)-scoring the highest P-value ( Fig 3A and Table S1). Exportin-2 mediates importin-alpha re-export from the nucleus to the cytoplasm, after NLS-containing import cargos have been released into the nucleoplasm, thus acting as a general regulator of nuclear import (36). The observed correlation suggests that AURKA overexpression in cancer associates with conditions of increased protein import. Interestingly, a similar correlation was observed for CSE1L and TPX2 (Fig 3A and Table S1). Indeed, AURKA and TPX2 are among the top five genes in the whole human genome most highly co-expressed in tumors together with CSE1L, with AURKA being the top-ranking one ( Fig 3B). Based on these results on the well-known nuclear localization of TPX2 in interphase (37,38) and its frequent co-overexpression with AURKA in tumors (references 39, 40; see heatmap profiles and tissue-wise expression in different cancer types compared with normal tissues in Fig 3C and D), we hypothesized that TPX2 influences the AurkA nuclear pool and that it may directly contribute to the increased nuclear localization of the kinase observed in certain cancer types (22).

TPX2 contributes to the accumulation of nuclear AurkA
To test this possibility, we first evaluated the correlation between AurkA and TPX2 levels in nuclei of non-transformed hTERT RPE-1 cells. A positive correlation of IF nuclear signals indicated that AurkA nuclear localization occurs in cells with high levels of TPX2 ( Fig 4A). Consistently, we noticed that the nuclear enrichment of AurkA observed in MG132-treated cells was accompanied by an accumulation of TPX2 ( Fig 2C). Interestingly, we revealed AurkA/ TPX2 in situ proximity ligation assay (isPLA) spots in nuclei of about 25% of interphases ( Fig 4B) consistent with the percentage of cells displaying nuclear AurkA ( Fig 1D) and indicating for the first time a nuclear interaction between the two proteins. To directly evaluate whether co-overexpression of TPX2 is able to increase the fraction of cells with nuclear-localized AurkA, we generated an additional hTERT RPE-1 cell line where both AURKA and TPX2 are overexpressed, in an inducible manner (Figs 4C and S2A). Both the percentage of cells displaying AurkA/TPX2 isPLA spots and the isPLA signal intensity significantly increase in the AURKA/TPX2-overexpressing cell line ( Fig 4B). Importantly, after dox induction 60% of AURKA/TPX2-overexpressing interphases are AurkA-positive, and AurkA is enriched in cell nuclei in about 40% of interphases ( Fig 4D), a fraction significantly higher compared with control and AURKA-overexpressing cell lines (see Figs 1 and 2). Treatment with LMB had no effect on either nuclear localization or AurkA levels (Figs 4D and S2B), supporting a key role of TPX2 in regulating the AurkA nuclear fraction. Again, when we treated these cells with MG132, both AurkA and TPX2 levels increased (Fig S2B) and the almost totality of cells (<90%) displayed nuclear AurkA ( Fig 4D). As for the AURKA-overexpressing cell line, treatment with epoxomicin yielded comparable results (Fig S2C).
To further assess the importance of the AurkA/TPX2 interaction, we used mutant/truncated versions of the two proteins. First, we analyzed the localization of the Venus-tagged AurkA S155R mutant, described to have an impaired interaction with TPX2 (41, 42) after transient transfection in U2OS cells, and observed a decreased nucleo/cytoplasmic ratio compared with AurkA WT signal (Figs 4E and S2D). We then evaluated how AurkA localizes after co-overexpression in hTERT RPE-1 with a truncated version of TPX2 lacking the AurkA interaction region (aa 1-43 (11,43)). To this aim, we generated an additional cell line for the stable and inducible cooverexpression of AURKA and TPX2Δ43 (Fig S2E). In this cell line, interphases with AurkA nuclear localization are comparable to control cultures and to the AURKA-overexpressing cell line ( Fig 4F). Consistently, cells with AurkA cytoplasmic localization (nuclearexcluded) represent the majority of AurkA-positive interphases in the AURKA alone and AURKA/TPX2Δ43 cell lines, whereas they almost disappear (about 10% of the population) in the AURKA/TPX2 cell line (compare Figs 2B and 4D and F). Furthermore, only in the AURKA/TPX2overexpressing cell line we observed an increase in PCNA-positive (i.e., Sphase) cells within the nuclear AurkA-positive population (Fig 4G).
Given the activating role of TPX2 on AurkA, we evaluated whether the catalytic activity of AurkA is required for its nuclear localization. We used 250 nM MLN8237 for 2 h to inhibit AurkA kinase activity ( Fig  4H). The percentage of cells displaying AurkA nuclear localization in control hTERT RPE-1 or AURKA/TPX2-overexpressing cultures did not change upon MLN8237 treatment (compared with control cultures), both in asynchronous and in G2-enriched (RO3306-treated) cultures. To confirm that AurkA kinase activity is not required for its nuclear localization, we transfected in U2OS cells a kinase-deficient mutant (AurkA K162R) and showed that its nucleus/cytoplasm ratio is comparable to that of AurkA WT (Fig 4I).
Together, these results indicate an important function of TPX2 in regulation of AurkA interphase localization and suggest that it may play a role, independent of its activating function, in nuclear enrichment of the kinase in cancer.  TPX2 promotes nuclear AurkA functions in a 3D breast cell-derived model In order to evaluate whether TPX2 co-overexpression has functional consequences on processes described to be dependent on nuclear AurkA, we generated additional cell lines for the expression of AURKA, or AURKA/TPX2, in MCF10A breast non-transformed epithelial cells (Fig 5), based on the ongoing active investigation of the nuclear role of AurkA in breast cancer (see the Introduction section). We first confirmed the localization of AurkA, which is mostly nuclear-excluded in cells overexpressing AurkA alone (Fig 5A, central panels) and became mainly nuclear when AurkA is cooverexpressed with TPX2 ( Fig 5A, right panels). It is well known that nuclear functions of AurkA correlate with an increase in transformed cells with breast cancer stem cell properties (24). Sphere-forming efficiency (SFE) and sphere size are used to assess the number of mammary cells with stem and EMT features, with SFE ranging from 0.1% to 0.7% in normal mammary epithelial cells, and from 1% to 3% in breast cancer cell lines (44,45). Therefore, we moved to a mammosphere 3D condition, where we confirmed similar levels of AurkA in both AURKA-and AURKA/TPX2-overexpressing MCF10A cell lines by WB ( Fig 5B) and RT-PCR ( Fig 5C). We evaluated their sphereforming ability by both counting the number and measuring the size of the obtained mammospheres. Both analyses demonstrate that AURKA overexpression increases mammosphere formation ( Fig 5D); importantly, the effects are exacerbated when TPX2 is co-overexpressed. Accordingly, the stemness marker CD44, previously shown to increase upon nuclear AURKA overexpression (24), is up-regulated in AURKAoverexpressing conditions, and even more in AURKA/TPX2-cooverexpressing mammospheres (Fig 5E), supporting the relevance of TPX2 as a determinant of AurkA nuclear functions. As an independent measure of AurkA nuclear functions, we evaluated the expression of one of the reported up-regulated genes of the hypoxia signaling signature (27), that is, CXCR4, by RT-PCR in the mammospheres (Fig 5E). Consistent with our hypothesis, AURKA/TPX2 overexpression leads to a significant increase in mRNA expression with respect to AURKA overexpression alone. Overall, these data support the idea that overexpressed TPX2 plays a key role in mediating the oncogenic functions previously described for nuclear AurkA (24,27).

Discussion
In the present work, we studied the mechanisms that regulate AurkA localization in interphase nuclei, a condition that is associated with its oncogenic properties (22). An increased AurkA nuclear localization is reported in the literature as a prognostic marker for poor survival, particularly in breast cancer (23). Emerging evidence proposes that this AurkA fraction operates through both kinase-dependent and kinase-independent roles in cell transformation and cancer (24,27), highlighting the importance to better understand its regulation.
Starting from physiological conditions, we report that nuclear AurkA localization becomes evident in G2 cells, about 90 min before mitotic entry, similar to what has been reported for exogenous AurkA (46). Interestingly, our data indicate a strong correlation between AurkA nuclear localization and high levels of its major regulator TPX2, which is nuclear in interphase (37). Most importantly, we revealed for the first time a nuclear interaction between AurkA and TPX2, in addition to the well-characterized interaction at spindle microtubules in mitosis (9), suggesting that TPX2 may be involved in the regulation of AurkA localization not only in mitosis, but also in G2 (Fig 5F). The interaction between TPX2 and AurkA in the nucleus may be a step required for correct mitotic entry. Interestingly, we recently reported that TPX2 and the spindle orientation regulator NuMA interact in interphase nuclei and at spindle poles (29), supporting the hypothesis that mitotic complexes start assembling in G2. An additional, not mutually exclusive, possibility is that the interaction between AurkA and TPX2 detected in interphase nuclei is linked to their emerging non-mitotic roles (19,21,38,47).
Despite the increasing interest that nuclear localization of AurkA is raising, no canonical NLS or NES sequences within the kinase, nor post-translational modifications related to interphase localization, have been described. It has been reported that the nuclear localization determinants are within the C-terminus, whereas the N-terminus contains the cytoplasmic ones (24). Indeed, Rannou and colleagues proposed that overexpressed AurkA undergoes active export (28). Consistently, we observed that inhibiting CRM1 increases the fraction of cells with nuclear AurkA in a cell cycle window that precedes G2, suggesting that AurkA displays a shuttling behavior in the S phase that prevents premature activation of mitotic pathways.
Given the emerging importance of nuclear AurkA in tumorigenesis, we then asked whether increasing its expression directly correlates with this localization pattern. Surprisingly, we observed that under overexpression conditions in non-transformed cells AurkA is mostly cytoplasmic and excluded from nuclei. Thus, tumors where AURKA is overexpressed and nuclear may have concomitant alterations in pathways that control AurkA nucleus/cytoplasm trafficking. Our analyses of gene expression databases identify AURKA as the gene with the highest correlation with CSE1L in multiple cancer types, with breast invasive carcinoma data displaying the highest significance. CSE1L mediates the re-export from the nucleus to the cytoplasm of importin-alpha, the major import vector together with importin-beta for NLS-containing proteins, ensuring efficient recycling and nuclear import (36). Our results suggest therefore that in certain cancer types, AURKA overexpression correlates with increased import rates. Interestingly, the association data are similar for TPX2, which we propose as highly relevant for enriched AurkA nuclear localization (see below), and the three genomic loci (AURKA, TPX2, and CSE1L) are all in the 20q chromosome arm, frequently amplified in cancer.
Inhibiting nuclear export by LMB treatment modified the localization pattern of overexpressed AurkA; surprisingly, the treatment concomitantly reduced protein levels of the kinase. This latter effect was counteracted by proteasome inhibition, which was able intensity (nucleus/cytoplasm ratio) (at least 230 cells per condition; three independent experiments). Error bars: SD; r: correlation value; ns, not significant; and ***P < 0.0001, chi-squared test  per se to induce a strong accumulation of AurkA in virtually all interphase nuclei. These data suggest that premature entry of AurkA in the nucleus-when overexpressed-is prevented, in cell cycle phases when AURKA is normally not expressed, by a proteasome-dependent mechanism (Fig 5F). Of note, the ubiquitin ligase involved in AurkA degradation at mitotic exit-APC/C Cdh1/Fzr1 -has been reported as nuclear and is active until the G1/S transition (32,33). Consistently, using inhibitors of APC/C Cdh1/Fzr1 we observed an increase in AURKA-overexpressing cells displaying nuclear localization. Thus, concomitant deregulation of ubiquitin ligases may be a route to increased levels of overexpressed AurkA in interphase nuclei. Evidence of lowered levels of Cdh1 in tumors, and specifically in breast cancer, exists (48,49). Additional ubiquitin ligases may contribute to this interphase modulation. In this respect, it is interesting to note that ubiquitin ligases that modulate AurkA stability, for example, Fbxw7 and VHL, are frequently mutated in cancer (references 22, 50 and references therein). It will be interesting to further investigate the connection between AurkA protein stabilization and cancer, initially proposed when constitutive stabilizing phosphorylation of Ser51 was shown in head and neck cancer (51), in light of the contribution to its nuclear-localized oncogenic functions. The AurkA nuclear enrichment observed upon proteasome inhibition may partially be the result of the stabilization of other factors, influencing in turn AurkA nuclear localization. TPX2 may be one such factor, because we observe that its levels increase after treatment with MG132. Given the frequent co-overexpression with AURKA in cancer (references 39, 52; updated data in Fig 3 of this  study), TPX2 is therefore an interesting candidate to promote nuclear AurkA oncogenic functions. Indeed, our results indicate that TPX2 co-overexpression is able to increase the nuclear localization of AurkA, in a manner that depends on its ability to bind AurkA ( Fig  5F). This effect does not require AurkA activity, thus not relying on the TPX2-activating function on AurkA. Further investigation will be required to clarify whether TPX2 is necessary for AurkA import into the nucleus, or whether it contributes to the nuclear accumulation of the kinase by binding it into the nucleus and protecting it from proteasome-dependent degradation and/or nuclear export. These results open the interesting possibility that co-overexpression of AURKA and TPX2 in cancer (39,40) is associated not only with altered mitotic functions resulting in chromosomal instability, but also with emerging nuclear functions of AurkA. Among these, it has been shown that nuclear AurkA promotes mammosphere formation and transcriptional activation of myc-and FOXM1-regulated genes promoting a stemness phenotype, and of a hypoxia network signature (24,25,27). Supporting our hypothesis, MCF10A breastderived cells engineered to overexpress AURKA and TPX2 yield an increase in the number and size of mammospheres, and transcriptional activation of stemness (CD44) or hypoxia signaling (CXCR4) genes, compared with overexpression of AURKA alone. Still, the latter situation is already associated with a lower but significant increase compared with control conditions. This may be due to the fraction of cells displaying nuclear AurkA when overexpressed alone. In an alternative scenario, given the pleiotropic roles of AurkA (19) there may be additional routes, not associated with the nuclear fraction of AurkA, cooperating to generate a stemness phenotype.
Our observations that TPX2 contributes to nuclear oncogenic functions of AurkA, described at least in part as non-kinasic, also open interesting opportunities in the therapeutic field. Although targeting AurkA was initially attempted by developing ATPcompetitive inhibitors, an approach that is still actively pursued (15), recent strategies include developing protein degraders (53,54) and protein-protein interaction inhibitors (55,56,57,58). Among the latter class, molecules that impair the formation of the AurkA/TPX2 complex may result in promising tools to disrupt AurkA nuclear functions in cancer, which would instead remain largely untargeted by molecules inhibiting its kinase activity.
In conclusion, understanding the regulation of nuclear AurkA may not only provide important information on its oncogenic functions but also drive the design of novel approaches to effectively target this pool of the kinase in specific tumor types where it has been shown to be relevant for cancer progression.

Materials and Methods
Cell cultures, synchronization protocols, and treatments normalize data). Error bars: SD; *P < 0.01; **P < 0.001; and ***P < 0.0001, t test (C, D, E) or Kruskal-Wallis test (D). Scale bars: 10 μm (A) and 100 μm (D). (F) Schematic model of the mechanisms that regulate AurkA localization in interphase nuclei. In control cells (top), AurkA nuclear accumulation in the S phase is prevented by the nuclear export protein CRM1; proteasome-dependent degradation may also contribute. In G2 cells, the interaction between AurkA and TPX2 promotes AurkA nuclear localization. Under AurkA overexpression conditions (AurkA OE, middle panels), AurkA nuclear accumulation in the G1 and S phases is limited by the action of APC/C Cdh1/Fzr1 and other ubiquitin ligases. These effects are counteracted in cells that overexpress both AurkA and TPX2 (AurkA/TPX2 OE, lower panels), by their interaction, thus promoting the accumulation of nuclear AurkA. The blue arrow represents cell cycle progression, and the two colored lines represent AurkA mRNA levels (exogenous: green; endogenous: purple). Created with BioRender.

Mammosphere cultures
Single-cell suspensions of MCF10A (and derived; see below) cell lines were grown in ultralow attachment six-well plates (Corning) at a density of 4,000 cells/ml as described in reference 60. After 10 d, the diameters of mammospheres were measured in phase-contrast pictures (ZOE) using ImageJ software. Numbers of mammospheres (diameter >50 µm) were counted, and the efficiency of mammosphere formation was evaluated (%SFE = number of mammospheres/number of plated cells * 100). The mammosphere pellet was collected by gentle centrifugation (300g, 5 min) for RNA and protein extraction.

Real-time PCR
Total RNA was extracted and reverse-transcribed as described in reference 60. Primers were designed as follows: Relative quantification was performed by the comparative cycle threshold method (61). The mRNA expression values were normalized to those of the TBP (TATA-box-binding protein) gene used as an endogenous control. One control of MCF10-derived mammospheres infected with the control vector (PLV) was randomly chosen as a control calibrator.

Generation of stable cell lines
Cell lines for the stable and inducible overexpression of myctagged Aurora-A in combination with either FLAG-TPX2 or FLAG-Δ43TPX2 were generated using the previously described procedure (62) and plasmids listed below.

Plasmids and transient transfections
Plasmids (epB_BSD_TT_Aurora-A-myc; epB_Puro_TT_FLAG-TPX2; epB_Puro_TT_FLAG-Δ43TPX2; and the hyPB7 plasmid encoding the hyperactive PiggyBac transposase) used for the generation of the stable transgenic hTERT RPE-1 cell lines were previously described (29,62,63). pVenus-N1-AURKA WT and mutant versions are previously published plasmids (54), with all cloning details available on request. Transient transfections of pVenus-N1-AURKA plasmids were carried out using a Neon electroporator (Thermo Fisher Scientific) according to the manufacturer's recommendations, and transfected cells were seeded onto eight-well microscope slides (Ibidi) for live-cell fluorescence microscopy.
pBK-CMV-myc-Aurora-A and pBK-CMV-myc-Aurora-A-K162R (kind gift of EA Nigg (64)) plasmids were transiently transfected in U2OS cells using Lipofectamine 2000 (Invitrogen Corporation, Thermo Fisher Scientific) following the manufacturer's instructions. The pBK-CMV empty vector was transfected in control cultures. Cells were harvested 30 h after transfection and analyzed by IF.
isPLA isPLAs were performed on cells grown on coverslips and fixed with formaldehyde (see the Immunofluorescence section) using the Duolink PLA kit (DUO92007 or DUO92008; Sigma-Aldrich) according to the manufacturer's instructions. The amplification time has been set to 60 min. The primary antibody pair to detect the interaction was mouse anti-Aurora-A/rabbit anti-TPX2, and DNA was stained with DAPI (see the Immunofluorescence section for details and acquisition procedures).

Time-lapse video recording
Time-lapse imaging was carried out using cells seeded onto Ibidi eight-well slides and imaged at 37°C in L-15 medium/10% FBS using a 40X N.A. 1.3 oil immersion objective. Images were acquired on an automated epifluorescence imaging platform consisting of an Olympus IX81 inverted microscope (Olympus Life Science) equipped with a LED illumination source and a motorized stage and fitted with an incubation chamber. Image acquisition was controlled by Micro-Manager (66) and images were exported as tiff files.

Image analysis and quantification
Images in Figs 2E and 4E were analyzed in ImageJ, with net green intensity (after background subtraction) in ROIs of 25 pixel radius from the nucleus and the cytoplasm used to calculate the nucleus/ cytoplasm ratio of fluorescence.
Images in Figs 2B and 4A and B were quantified as follows: the "general analysis" module of NIS-Elements H.C. 5.11 was used for automatic recognition of the nuclei defined by the DAPI signal in all images. Nuclear signal intensity for AurkA, TPX2, or AurkA/TPX2 isPLA was then measured on maximum intensity projections from acquired z-stacks, after correction for external background. The AurkA-myc nucleus/cytoplasm ratio of fluorescence was calculated from the average values of signals within the nucleus and a cytoplasmic ROI. A threshold to automatically identify positive cells (isPLA or AURKA WT/K162R-transfected) was set at two standard deviations above the mean of the negative control conditions (isPLA negative control or PBK empty vector-transfected cultures).

FACS analysis
For FACS analysis, cells were collected by centrifugation, washed with PBS, and fixed with 50% methanol overnight at 4°C. Cell cycle phase distribution was analyzed after incubation for 30 min in the dark with propidium iodide (PI, P4170, 0.03 mg/ml; Sigma-Aldrich) and RNase A (0.2 mg/ml) using a flow cytofluorometric (BD FACS-Calibur) apparatus. Cell debrides and aggregates were gated out on the biparametric graph FL2-A/FL2-W. At least 15,000 events per sample were acquired. The percentage of cells in the different phases of the cell cycle was calculated using Floreada.io software.

Expression analysis
The mRNA expression of AURKA, TPX2, and their co-expressed genes in cancers was obtained using Gene Expression Profiling Interactive Analysis API, to request the expression analysis from 9,736 tumors and 8,587 normal samples from TCGA and the GTEx data projects (67). Proteins involved in nucleus/cytoplasm shuttling, with expression patterns similar to AURKA and TPX2 across different tumors, were selected from GO (GO: 0006611, 65 genes; GO: 0006606, 177 genes). Pairwise expression correlation was assessed using Pearson's correlation analysis.

Statistical analyses
Data were statistically analyzed using the InStat3 GraphPad 7 by (i) unpaired t tests and ordinary one-way ANOVA multiple comparison tests for measurements of continuous variables; when samples were not normally distributed, the Mann-Whitney and Kruskal-Wallis tests, respectively, were used instead; (ii) chi-squared (and Fisher's exact) tests, in the contingency table analyses for measurements of categorical variables; and (iii) Spearman's (non-normally distributed samples) correlation for single-cell correlation analysis. The number of replicates and sample size are indicated in the corresponding figure legends. The criterion for statistical significance (*) was set at P < 0.01. C Lindon: conceptualization, supervision, funding acquisition, and writing-review and editing. G Guarguaglini: conceptualization, supervision, funding acquisition, project administration, and writing-original draft, review, and editing.